ABSTRACTMonocytes and macrophages are part of the first-line defense against bacterial, fungal, and viral infections during host immune responses; they express high levels of proinflammatory cytokines and cytotoxic molecules, including nitric oxide, reactive oxygen species, and their reaction product peroxynitrite. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death. Honey, in addition to its well-known sweetening properties, is a natural antioxidant that has been used since ancient times in traditional medicine. We examined the ability of Gelam honey, derived from the Gelam tree (Melaleuca spp.), to scavenge peroxynitrite during immune responses mounted in the murine macrophage cell line RAW 264.7 when stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ) and in LPS-treated rats. Gelam honey significantly improved the viability of LPS/IFN-γ-treated RAW 264.7 cells and inhibited nitric oxide production-similar to the effects observed with an inhibitor of inducible nitric oxide synthase (1400W). Furthermore, honey, but not 1400W, inhibited peroxynitrite production from the synthetic substrate 3-morpholinosydnonimine (SIN-1) and prevented the peroxynitrite-mediated conversion of dihydrorhodamine 123 to its fluorescent oxidation product rhodamine 123. Honey inhibited peroxynitrite synthesis in LPS-treated rats. Thus, honey may attenuate inflammatory responses that lead to cell damage and death, suggesting its therapeutic uses for several inflammatory disorders.

f4-ijms-13-12113: Effect of honey on the peroxynitrite-induced oxidation of DHR-123. (A) SIN-1, a peroxynitrite donor, was incubated for 2 h with different dilutions of honey (in PBS), 1400W (100 μM), and DHR-123, and the formation of rhodamine 123 was measured; (B) Honey (different dilutions in PBS) or 1400W (100 μM) were incubated for 15 min with DHR-123 and peroxynitrite, and the formation of rhodamine 123 was measured; (C) RAW 264.7 cells were incubated with honey (different dilutions in PBS), 1400W (100 μM), and DHR-123 for 60 min. Then, LPS/IFN-γ was added, and the cultures were incubated for an additional 24 h, after which the formation of rhodamine 123 was measured. Results are expressed as a percentage of the control (mean ± SEM of 3 independent experiments performed in triplicate). *** p < 0.001 and ** p < 0.003. Rhodamine 123 formation in the untreated control was defined as 100%.

Mentions:
An increase in the oxidation of dihydrorhodamine 123 (DHR-123) is indicative of the presence of peroxynitrite. In the 3-morpholinosydnonimine (SIN-1) model, honey was found to be a potent scavenger of peroxynitrite, inhibiting the SIN-1–induced oxidation of DHR-123 to rhodamine 123 with a half-maximal inhibitory concentration (IC50) of 0.148 mg/mL. Highly significant levels of oxidation were recorded in the presence of the iNOS inhibitor 1400W and in the (untreated) positive control (Figure 4A). To directly confirm the peroxynitrite scavenging activity of honey, peroxynitrite was incubated with or without different concentrations of honey (Figure 4B). In these experiments, honey also inhibited DHR-123 oxidation, presumably by scavenging peroxynitrite, with an IC50 of 0.68 mg/mL. However, oxidation was not inhibited by 1400W because the level of rhodamine 123 fluorescence was the same as that in the untreated control (Figure 4B). The addition of Gelam honey to RAW 264.7 cells that were induced with LPS/IFN-γ for 24 h also completely attenuated peroxynitrite activity and had an IC50 of 0.254 mg/mL (Figure 4C). In this experiment (unlike the previous 2 experiments), peroxynitrite synthesis was inhibited, and DHR-123 was not converted to its fluorescent product because cellular iNOS was blocked by 1400W (100 μM) (Figure 4C).

f4-ijms-13-12113: Effect of honey on the peroxynitrite-induced oxidation of DHR-123. (A) SIN-1, a peroxynitrite donor, was incubated for 2 h with different dilutions of honey (in PBS), 1400W (100 μM), and DHR-123, and the formation of rhodamine 123 was measured; (B) Honey (different dilutions in PBS) or 1400W (100 μM) were incubated for 15 min with DHR-123 and peroxynitrite, and the formation of rhodamine 123 was measured; (C) RAW 264.7 cells were incubated with honey (different dilutions in PBS), 1400W (100 μM), and DHR-123 for 60 min. Then, LPS/IFN-γ was added, and the cultures were incubated for an additional 24 h, after which the formation of rhodamine 123 was measured. Results are expressed as a percentage of the control (mean ± SEM of 3 independent experiments performed in triplicate). *** p < 0.001 and ** p < 0.003. Rhodamine 123 formation in the untreated control was defined as 100%.

Mentions:
An increase in the oxidation of dihydrorhodamine 123 (DHR-123) is indicative of the presence of peroxynitrite. In the 3-morpholinosydnonimine (SIN-1) model, honey was found to be a potent scavenger of peroxynitrite, inhibiting the SIN-1–induced oxidation of DHR-123 to rhodamine 123 with a half-maximal inhibitory concentration (IC50) of 0.148 mg/mL. Highly significant levels of oxidation were recorded in the presence of the iNOS inhibitor 1400W and in the (untreated) positive control (Figure 4A). To directly confirm the peroxynitrite scavenging activity of honey, peroxynitrite was incubated with or without different concentrations of honey (Figure 4B). In these experiments, honey also inhibited DHR-123 oxidation, presumably by scavenging peroxynitrite, with an IC50 of 0.68 mg/mL. However, oxidation was not inhibited by 1400W because the level of rhodamine 123 fluorescence was the same as that in the untreated control (Figure 4B). The addition of Gelam honey to RAW 264.7 cells that were induced with LPS/IFN-γ for 24 h also completely attenuated peroxynitrite activity and had an IC50 of 0.254 mg/mL (Figure 4C). In this experiment (unlike the previous 2 experiments), peroxynitrite synthesis was inhibited, and DHR-123 was not converted to its fluorescent product because cellular iNOS was blocked by 1400W (100 μM) (Figure 4C).

ABSTRACTMonocytes and macrophages are part of the first-line defense against bacterial, fungal, and viral infections during host immune responses; they express high levels of proinflammatory cytokines and cytotoxic molecules, including nitric oxide, reactive oxygen species, and their reaction product peroxynitrite. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death. Honey, in addition to its well-known sweetening properties, is a natural antioxidant that has been used since ancient times in traditional medicine. We examined the ability of Gelam honey, derived from the Gelam tree (Melaleuca spp.), to scavenge peroxynitrite during immune responses mounted in the murine macrophage cell line RAW 264.7 when stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ) and in LPS-treated rats. Gelam honey significantly improved the viability of LPS/IFN-γ-treated RAW 264.7 cells and inhibited nitric oxide production-similar to the effects observed with an inhibitor of inducible nitric oxide synthase (1400W). Furthermore, honey, but not 1400W, inhibited peroxynitrite production from the synthetic substrate 3-morpholinosydnonimine (SIN-1) and prevented the peroxynitrite-mediated conversion of dihydrorhodamine 123 to its fluorescent oxidation product rhodamine 123. Honey inhibited peroxynitrite synthesis in LPS-treated rats. Thus, honey may attenuate inflammatory responses that lead to cell damage and death, suggesting its therapeutic uses for several inflammatory disorders.